To continue the molecular analyzes, the DNA must be extracted from the tissues which were gathered during the Santo 2006 project. Here is a small summary of the technique used, with the description of the action of the chemical agents used at each step. Even if the techniques differ, the steps presented here are the same as those of the extraction protocol carried out with high school students.
The SANTO 2006 notably made it possible to collect many alcohol-preserved specimens so that they could subsequently study their DNA. As alcohol helps preserve DNA by replacing the water contained in cells, which has the effect of inhibiting enzymes that could lyse macromolecules, including DNA. However, DNA is not directly accessible and analyzed. The tissues must, therefore, undergo a series of treatments, the objective of which is to isolate the DNA contained in the cells, from all the other elements present in these same cells.
This technique, called "DNA extraction," is commonly used in many scientific fields involved in working with DNA. In evolutionary biology, phylogeny and population genetics, molecular approaches often require "reading" DNA sequences in order to infer relationships between and within species. Here, we explain the different stages of DNA extraction, specifying at each stage the different existing methods. These steps are, moreover, identical to that used for the extraction of DNA proposed for the second class, even if the method and the products used may vary.
In prokaryotes (bacteria and archaebacteria), DNA is simply contained in the cell, without further compartmentalization. On the other hand, in eukaryotes, which include all the specimens collected in Santo, DNA is contained by three types of compartments inside cells. Thus, to access DNA, the cell membrane and another membrane (nuclear, mitochondrial, or chloroplastic depending on the case) must be crossed. In addition, in multicellular organisms, cells are organized into tissue that must be dissociated to access DNA.
1: cell membrane, 2: nucleus, 3: mitochondria
At the molecular level, DNA is more or less directly associated with all kinds of protein, carbohydrate, and nucleic molecules. These interactions can be particularly strong, as, for example, with histones, proteins which allow DNA to wind. Thus, DNA is most of the time in the compacted form (except during events particularly in the life of a cell, such as a mitosis or meiosis). Other molecules interact with DNA, like other proteins and nucleic acids, linked to the regulation of gene expression, the duplication of DNA, its transcription into RNA, etc.
DNA wraps around itself, then around small protein beads (histones) to end up in the compacted form in the cell.
The objective of the extraction is therefore to isolate the DNA molecule, that is to say, separate it from all the other constituents of a tissue, including molecules strongly linked to DNA, and to '' obtain a sample that is sufficiently pure and in sufficient quantity to allow all the molecular biology manipulations linked to phylogeny and population genetics. Good tissue preservation is therefore essential for the DNA of a specimen that is too old may have been degraded, which will make it difficult to extract.
The pieces of tissue, taken directly from the collected specimens, must first be fragmented, to dissociate the tissues, cell walls, intracellular membranes, and the proteins that surround the DNA. A first mechanical step may be necessary, as in the case of arthropods where the tissues are inside a chitinous "shell." This can be done using a machine; a rapid and repeated lateral movement causes the displacement of a metal ball placed in the tube containing the tissue, which has the effect of crushing the tissues. In a second step, the tissues are placed in a buffer solution which contains, in particular, a cleanser, which has the effect of dissociating the membranes (of lipid nature, they are attacked in the same way as dishwashing liquid attacks fats), and an enzyme, proteinase, activated at 56 ° C. During this stage which can last several hours, the enzyme will "digest" the proteins contained in the cell, and in particular those which are linked to DNA.
The DNA jellyfish observed at the end of the experiment, therefore, contains DNA and the proteins associated with it. These proteins are problematic, for example, when the DNA is sequenced. The techniques we use, therefore making it possible, in addition to eliminating these proteins, to allow access to DNA.
The DNA is, therefore, no longer associated with the other constituents of the cells but remains mixed with them in the extraction buffer. To separate DNA, several methods exist:
- different chemical agents can make it possible to separate the DNA from the other constituents, for example, by obtaining 2 phases by adding Chloroform Iso-Amyl (CIA) and centrifugation, one which contains the DNA and the other the residues which one wishes eliminate. The DNA is then precipitated, adding, for example, NaCl (Sodium Chloride). It is by this process that DNA becomes visible in the form of a “jellyfish.”
- The solution can be passed through a positively charged column, which acts as a filter by retaining the DNA (negatively charged) and letting the other constituents pass.
Again, depending on the method used in the previous step, there are several ways to recover DNA after cleaning:
- After adding the different products to clean the DNA, the tube contains the solution, and the DNA is centrifuged. The DNA is then found in the form of a pellet, which is to say a solid precipitate bonded to the bottom of the tube, which contains the buffer. Then simply remove the buffer without dropping the pellet, and then allow the DNA to dry. The dried DNA is then eluted in a suitable buffer to avoid its degradation.
- The DNA attached to the column is then "unhooked" by adding a solution that reverses the relationships of the affinity of the column for DNA. As with the other method, the DNA is eluted in a suitable buffer.